Toxic Protein Expression

Expression of heterologous proteins presents many challenges. In E. coli, expression of a non-native protein often adversely affects the viability of the host cell both during the transformation stage and during protein expression. To improve host viability and consequently improve the potential for target protein over-expression, well-regulated expression systems should be employed. In E. coli expression systems, regulation is often provided by the host cell as well as the expression vector. IPTG-inducible systems rely on the Lac repressor to bind to lac, tac, trc or T7-lac promoters to inhibit expression until the culture reaches an optimal density for induction. In T7 systems, the Lac repressor is also important but an even more effective means to control expression is to employ a host strain that expresses a T7 RNA polymerase inhibitor protein (e.g. LysY).

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Usage Guidelines

A T7 expression plasmid containing a gene encoding E. coli UDG was transformed into each host, grown to 0.6 OD and induced for 3 hours. Comparison of soluble extracts from uninduced (-) and induced (+) cells shows superior control of expression in the T7 Express hosts while maintaining high levels of induced expression.

Disulfide bond formation in the cytoplasm of wild type E. coli is not favorable, while SHuffle is capable of correctly folding proteins with multiple disulfide bonds in the cytoplasm.

PfCHT1 Chitinase Expression in SHuffle® T7 Express

Plasmodium falciparum chitinase (PfCHT1) with three cysteines was expressed from a plasmid under the regulation of T7 promoter. After induction, cells were harvested and crude cell lysates were prepared. PfCHT1 was assayed using a chromogenic substrate (CalBioChem #474550) and standardized to protein concentration using Bradford reagent.

vtPA Expression in SHuffle®

Truncated tissue plasminogen activator (vtPA), which contains nine disulfide bonds when folded and oxidized correctly, was expressed from a pTrc99a plasmid in the cytoplasm of E. coli cells. After induction, cells were harvested and crude cell lysates were prepared. vtPA was assayed
using a chromogenic substrate Chromozym t-PA (Roche #11093037001) and standardized to protein concentration using Bradford reagent. E. coli wt + cells are DHB4, which is the parent of FÅ113 (Origami™).

Optimization of YidC-GFP Expression with Lemo21(DE3)

Protein expression with Lemo21(DE3) is very similar to BL21(DE3), with
only a few minor changes.

Protein Expression Using the PURExpress® In Vitro Protein Synthesis Kit

25 µl reactions containing 250 ng template DNA were incubated at 37°C for 2 hours. 2.5 µl of each reaction was analyzed by SDS-PAGE using a 10–20% Tris-glycine gel. Note that proteins can be purified using reverse affinity chromatography (reagents not supplied). The red dot indicates the protein of interest. Marker M is the Protein Ladder (NEB #P7703)

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